Purpose: A subset of head and neck cancers is associated with the human papillomavirus (HPV). Viral infection is closely correlated with expression of p16^INK4A in these tumors. We evaluated p16^INK4A as a prognostic marker of treatment response and survival in a well-defined and prospectively collected cohort of patients treated solely with conventional radiotherapy in the Danish Head and Neck Cancer Group (DAHANCA) 5 trial. Patients and Methods: Immunohistochemical expression of p16^INK4A was analyzed in pretreatment paraffin-embedded tumor blocks from 156 patients treated with conventional primary radiotherapy alone. The influence of p16^INK4A status on locoregional tumor control, disease-specific survival, and overall survival after radiotherapy was evaluated. Results: p16^INK4A positivity was found in 35 tumors (22%). Tumor-positivity for p16^INK4A was significantly correlated with improved locoregional tumor control (5-year actuarial values 58% v 28%; P = .0005), improved disease-specific survival (72% v 34%; P = .0006), and improved overall survival (62% v 26%; P = .0003). In multivariate analysis, p16^INK4A remained a strong independent prognostic factor for locoregional failure (hazard ratio [HR], 0.35; 95% CI, 0.19 to 0.64), disease-specific death (HR, 0.36; 95% CI, 0.20 to 0.64), and overall death (HR, 0.44; 95% CI, 0.28 to 0.68). Conclusion: Expression of p16^INK4A has a major impact on treatment response and survival in patients with head and neck cancer treated with conventional radiotherapy.

Objective To develop an affordable and robust pipeline for selection of patient-specific somatic structural variants (SSVs) being informative about radicality of the primary resection, response to adjuvant therapy, incipient recurrence and response to treatment performed in relation to diagnosis of recurrence. Design We have established efficient procedures for identification of SSVs by next-generation sequencing and subsequent quantification of 3–6 SSVs in plasma. The consequence of intratumour heterogeneity on our approach was assessed. The level of circulating tumour DNA (ctDNA) was quantified in 151 serial plasma samples from six relapsing and five non-relapsing colorectal cancer (CRC) patients by droplet digital PCR, and correlated to clinical findings. Results Up to six personalised assays were designed for each patient. Our approach enabled efficient temporal assessment of disease status, response to surgical and oncological intervention, and early detection of incipient recurrence. Our approach provided 2–15 (mean 10) months' lead time on detection of metastatic recurrence compared to conventional follow-up. The sensitivity and specificity of the SSVs in terms of detecting postsurgery relapse were 100%. Conclusions We show that assessment of ctDNA is a non-invasive, exquisitely specific and highly sensitive approach for monitoring disease load, which has the potential to provide clinically relevant lead times compared with conventional methods. Furthermore, we provide a low-coverage protocol optimised for identifying SSVs with excellent correlation between SSVs identified in tumours and matched metastases. Application of ctDNA analysis has the potential to change clinical practice in the management of CRC.

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5% success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70–80% of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.